Prenylcysteine oxidase 1 like protein is required for neutrophil bactericidal activities

The bactericidal function of neutrophils is dependent on a myriad of intrinsic and extrinsic stimuli. Using systems immunology approaches we identify microbiome- and infection-induced changes in neutrophils. We focus on investigating the Prenylcysteine oxidase 1 like (Pcyox1l) protein function. Murine and human Pcyox1l proteins share ninety four percent aminoacid homology revealing significant evolutionary conservation and implicating Pcyox1l in mediating important biological functions. Here we show that the loss of Pcyox1l protein results in significant reductions in the mevalonate pathway impacting autophagy and cellular viability under homeostatic conditions. Concurrently, Pcyox1l CRISPRed-out neutrophils exhibit deficient bactericidal properties. Pcyox1l knock-out mice demonstrate significant susceptibility to infection with the gram-negative pathogen Psuedomonas aeruginosa exemplified through increased neutrophil infiltrates, hemorrhaging, and reduced bactericidal functionality. Cumulatively, we ascribe a function to Pcyox1l protein in modulation of the prenylation pathway and suggest connections beween metabolic responses and neutrophil functionality.


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Sample size is described under each data set. All cohort predictions were based on Power G analysis to obtain significance level of p<0.05 at 80% probability.
Data exclusion is described in the Methods section. Animals who displayed wounding at sites different than the infection site were excluded. Animals that displayed infection-nonrelated opacification of either cornea were excluded.
Infections experiments with the P. aeruginosa clinical isolate 6294 were repeated twice and data are presented cumulatively. Infection experiments with the P. aeruginosa PAO1 strain was done once. Additional replication of the PAO1-induced infection were not carried out as the clinical isolate-induced infections recreated the phenotype of the lab-strain-induced infections.
The relative cohort sizes were small negating the use of randomization. Additionally, Pcyox1l KO mice displayed behavioural changes interfering with randomization.
CFU and pathology determinations were blinded.
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March 2021
Materials & experimental systems  The immortalized neutrophil progenitor cell lines are derived from male ER-Hoxb8 C57BL6/N mice.
The authentication of the KO cell lines was carried out by WB analysis.

Tested negative
No commonly misidentified cell lines were used in the study.
The Pcyox1l mouse line 49020 was a product of CRISPR targeting to zygotes, developed by the Knockout Mouse Production and Phenotyping project (KOMP2) at the Jackson Laboratory, and deposited to the MMRRC. Pcyox1l KO mice (line 49020) were cryorecovered and intercrossed to generate Pcyox1l KO and WT littermates for experiments. The Pcyox1l KO are on C57Bl6/J background. 6-9 week-old mice were used for experiments. Additionally, 6-9 week-old mice SPF and GF SW mice were used in the study. The CD18 KO breeders were purchased from Jackson Labs (line 002128). Mice were maintained as breeding pairs at MCP with 12h dark and 12 light cycles with 40-60% humidity and at 65 -75F. Sex as a biological variable was not considered as prior experiments in mice did not point to sex as a determinant in clinical outcomes of P. aeruginosa-induced keratitis. Similarly, there is no sex-bias in human disease severity.
No wild type mice were used in the study. No